Gemcitabine ProTide hypoxia-activated prodrug and application thereof

ABSTRACT

A gemcitabine ProTide hypoxic-activated prodrug and a use thereof in the preparation of a medicament for treating tumors. The general structural formula thereof is formula (A), wherein: one of R1 and R2 is a hypoxic-activated group of —C(R3R4)ArNO2, and the other is an alkyl group of 1 to 6 carbon atoms, a phenyl group or —CH2Ar, wherein R3 and R4 are —H or a methyl group, and —Ar is an aromatic ring compound. The gemcitabine ProTide hypoxic-activated prodrug described in the present invention has a stronger cytotoxicity under a hypoxic condition, has excellent anti-tumor effects and is very safe; the present invention can be used along with other anti-tumor drugs to exert a better anti-tumor activity, and can be used in the preparation of a medicament for treating tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase entry of InternationalApplication No. PCT/CN2017/095794, filed on Aug. 3, 2017, which is basedupon and claims priority to Chinese Patent Application No.201610649914.X, filed on Aug. 9, 2016, and Chinese Patent ApplicationNo. 201710610509.1, filed on Jul. 25, 2017, the entire contents of whichare incorporated herein by reference.

TECHNICAL FIELD

The present invention belongs to the field of pharmacy, and provides agemcitabine ProTide hypoxic-activated prodrug and a use thereof.

BACKGROUND

Gemcitabine is a nucleoside anti-tumor drug. The mechanism of action ofthis drug is to antagonize nucleotide metabolism. After intracellulartriphosphorylation in vivo, gemcitabine specifically interferes withnucleic acid metabolism and prevents cell division and reproduction byinhibiting the synthesis of deoxynucleoside triphosphate (dNTPs),interfering with cell replication by being incorporated into DNA or RNAmolecules, competitively inhibiting DNA polymerase, and the like, thuseventually causing the death of tumor cells. Nucleoside anti-tumor drugsare prone to drug resistance, but ProTide prodrugs thereof can reducethe occurrence of drug resistance and have a good anti-tumor effect,among which a gemcitabine ProTide prodrug NUC-1031 has been clinicallystudied (Journal of Medical Chemistry 2014, 57, 1531-1542). However,ProTide prodrugs cannot reduce the toxic and side effects of drugs onnon-tumor tissues.

With the rapid growth of tumors, some tumor tissues are farther andfarther away from the nearest blood vessel, and oxygen supply isinsufficient, resulting in tumor hypoxia (Nature review cancer 2002, 2:38-47). Traditional anti-tumor drugs have good lethality to tumors nearblood vessels, but have limited effects on tumors in hypoxic regions.Tumor hypoxic-activated prodrugs can specifically release anti-tumoractive constituents in tumor hypoxic regions, thus killing tumors in thehypoxic regions (Chinese Journal of Cancer 2014, 33: 80-86).Hypoxic-activated prodrugs have a tumor targeting property, thus havinga better safety performance, and a better anti-tumor effect when used incombination with traditional anti-tumor drugs, among which TH302 hasbeen clinically studied and has a good therapeutic effect on pancreaticcancer (Journal of Clinical Oncology 2015, 33, 1475-1482).

SUMMARY Technical Problem

The present invention provides a gemcitabine ProTide hypoxic-activatedprodrug and a use thereof. The prodrug has a stronger cytotoxicity undera hypoxic condition, has excellent anti-tumor effects and is very safe,can be used along with traditional anti-tumor drugs to exert a goodanti-tumor activity at a small dose, and can be used in the preparationof a medicament for treating tumors.

Technical Solution

The chemical structural formula of the gemcitabine ProTidehypoxic-activated prodrug is:

wherein one of R¹ and R² is a hypoxic-activated group of —C(R³R⁴)ArNO₂,the other is an alkyl group of 1 to 6 carbon atoms, a phenyl group or—CH₂Ar, R³ and R⁴ are —H or a methyl group, and —Ar is an aromatic ringcompound.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activatedprodrug, the structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ is —H or amethyl group, and R⁴ is a methyl group.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activatedprodrug, R¹ is a phenyl group, and the structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activatedprodrug, the structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ and R⁴ are —H.

As a preferred scheme, for the gemcitabine ProTide hypoxic-activatedprodrug, R¹ is —CH₂Ar, —Ar is a benzene ring with an electron donatinggroup, and the structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.

As a preferred scheme, the structure of the gemcitabine ProTidehypoxic-activated prodrug is as follows:

As a preferred scheme, the structure of the gemcitabine ProTidehypoxic-activated prodrug is as follows:

A use of the above compound or pharmaceutically acceptable salt thereofin the preparation of a medicament for treating tumors.

A use of a composition of the above compound or pharmaceuticallyacceptable salt thereof and gemcitabine hydrochloride in the preparationof a medicament for treating tumors.

A medicament for treating tumors, of which the effective component beingthe above gemcitabine ProTide hypoxic-activated prodrug orpharmaceutically acceptable salt thereof.

A medicament for treating tumors, of which the effective component beingthe composition of the above gemcitabine ProTide hypoxic-activatedprodrug or pharmaceutically acceptable salt thereof and gemcitabinehydrochloride.

It should be pointed out that our research found that the cytotoxicityof compounds 001-021 under a hypoxic condition was significantly higherthan that under a normal oxygen condition. The cytotoxicity of a targetcompound (e.g., compound 022) obtained by introducing ahypoxic-activated group into an amino position of gemcitabine ProTideprodrug NUC-1031 under a hypoxic condition was not significantlydifferent from that under a normal oxygen condition (see Table 1). Itindicated that the introduction position of the hypoxic-activated groupwas specific for maintaining the hypoxic-activated function of drugs.

It should also be pointed out that our research found that when R² was ahypoxic-activated group, if R³ and R⁴ were both H (such as compound023), the cytotoxicity of the target compound under a hypoxic conditionwas not significantly different from that under a normal oxygencondition (see Table 1), while when R¹ was a hypoxic-activated group, ifR³ and R⁴ were both H (such as compounds 013-017), the cytotoxicity ofthe target compound under a hypoxic condition was significantly higherthan that under a normal oxygen condition (see Table 1).

When R¹ was a hypoxic-activated group, R² was —CH₂Ar, and Ar was aphenyl group with an electron donating group, the target compound showeda stronger cytotoxicity under a hypoxic condition (such as compounds018-021); when Ar was a phenyl group having no substituent group orhaving an electron with-drawing group (such as compound 024), thecytotoxicity of the target compound under a hypoxic condition differedslightly from that under a normal oxygen condition.

Taking compound 001 as an example, the anti-tumor effect and safetyperformance of the target compound were investigated. The targetcompound showed a significant anti-tumor growth effect (see FIG. 1 andFIG. 2). At 4 times of treatment dose, compared with a control group,there was no significant difference in animal weight, indicating thatthe target compound had good safety performance (see FIG. 3). Combineduse with traditional anti-tumor drugs such as gemcitabine can generatebetter anti-tumor effect (see FIG. 2).

Advantageous Effect

The gemcitabine ProTide hypoxic-activated prodrug of the presentinvention has a small cytotoxicity in a normal oxygen environment andstrong cytotoxicity under a hypoxic condition, therefore, thegemcitabine ProTide hypoxic-activated prodrug can specifically play ananti-tumor effect on tumors in tumor hypoxic regions, reduce toxic andside effects on other tissues, has an excellent anti-cancer effect andgood safety performance, can be used together with traditionalanti-tumor drugs such as gemcitabine to generate a good anti-tumoreffect at a small dose, and can be used for preparing medicaments fortreating tumors. Further research finds that at 4 times of effectivedose, the gemcitabine ProTide hypoxic-activated prodrug provided by thepresent invention has no obvious toxic effect increase compared with lowdose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a growth inhibition effect of a targetcompound 001 on orthotopic transplantation tumor of human BxPC-3 nudemice. After administration, the pancreatic cancer tumor tissue qualityof nude mice in an experimental group (compound 001) was significantlylower than that of a gemcitabine group and an NUC-1031 group, indicatinga better tumor growth inhibition effect.

FIG. 2 is a schematic diagram of a growth inhibition effect of a targetcompound 001 on subcutaneous orthotopic transplantation tumor of humanBxPC-3 nude mice. After administration, the pancreatic cancer tumortissue volume of nude mice in a high-dose group and alow-dose+gemcitabine group was significantly lower than that of agemcitabine group, indicating a better tumor growth inhibition effect.

FIG. 3 is a schematic diagram of changes in animal weight when a targetcompound 001 is used for treating subcutaneous orthotopictransplantation tumor of human BxPC-3 nude mice. Compared with alow-dose group (1 time of the effective dose), the animal weight of ahigh-dose group (4 times of the effective dose) is not significantlydifferent from that of the low-dose group and a positive control druggemcitabine group, indicating that the target compound has good safetyperformance.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The following embodiments enable those skilled in the art to fullyunderstand the present invention, but do not limit the present inventionin any way.

Embodiment 1: Synthesis of Target Compounds 001-021

Synthesis of 3′-O-(t-butyloxycarboryl) gemcitabine

Synthesis route (method reference, The Journal of Organic Chemistry,1999, 64: 8319-8322):

Experimental operation: stir gemcitabine (0.60 g, 2 mmol), Na₂CO₃ (1.06g), 40 mL dioxane, 40 mL water, di-tert-butyl dicarbonate ester (DBDC,0.44 g, 2 mmol) at room temperature for 48 h; add 20 mL water, extractwith 2×300 mL ethyl acetate, dry with Na₂SO₄, and perform vacuumconcentration; perform flash column chromatography on (CH₂Cl₂-ethylacetate-EtOH 1:1:0.02) to obtain 3′-O—(N-t-butyloxycarboryl) gemcitabine(0.60 g). ¹H NMR (DMSO-d6, 300 MHz) δ(ppm): 7.64 (d, 1H) 7.40 (d, 2H),6.21 (t, 1H), 5.81 (d, 1H), 5.25-5.12 (m, 2H), 4.13 (t, 1H) 3.71-3.60(m, 2H), 1, 45 (s, 9H).

Synthesis route (method reference, The Journal of Medicinal Chemistry,2014, 57:1531-1542):

Add 10 mL redistilled dichloromethane into a 100 mL eggplant shapeflask, add phosphorus oxychloride (0.2 g, 1.3 mmol) intodichloromethane, place the reaction system at −78° C., add triethylamine(0.13 g, 1.3 mmol) therein, stir for 15 min, then dropwise add adichloromethane solution (5 mL) of phenol (0.153 g, 1 mmol), react at−78° C. for 1 h after 15 min of dropping, and react at room temperaturefor 1 h; leave the whole reaction system at −78° C., add 0.23 g (2.3mmol) triethylamine therein, stir for 15 min, add 10 ml redistilleddichloromethane solution containing 0.275 g (1 mmol)L-alanine-1-(4-nitrophenyl) ethanol ester hydrochloride, and stir for 3h; add 30 mL redistilled dichloromethane into another 50 mL eggplantshape flask, add 0.29 g (0.8 mmol) 3′-O-(t-butyloxycarboryl)gemcitabine, add 0.2 g (2 mmol) triethylamine, add 2 mLN-methylimidazole, and add the dichloromethane solution into the 100 mLreaction system in the previous flask at room temperature for overnightreaction at room temperature; concentrate the solvent, filter outinsoluble substances, wash the filtrate with 3×30 mL water, extract withdichloromethane, concentrate the solvent, stir with 6 mL trifluoroaceticacid and dichloromethane (volume ratio being 1:1) at 0° C. for 4 h,concentrate the solvent, and perform flash column chromatography (volumeratio of dichloromethane:methanol being 20:1); and obtain the compound001: ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.22-8.21 (m, 2H), 7.60-7.55 (m,1H), 7.52-7.51 (m, 2H), 7.41-7.36 (m, 2H), 7.29-7.22 (m, 3H), 6.30-6.25(m, 1H), 6.04-5.89 (m, 2H), 4.56-4.38 (m, 2H), 4.28-4.21 (m, 1H),4.15-4.10 (m, 1H), 3.97-3.91 (m, 1H), 1.56-1.55 (m, 3H), 1.38-1.32 (m,3H).

Compounds 002-021 were synthesized with the same method.

Referring to the method for producing the compound 001, phenol,L-alanine-1-(4-nitrophenyl)-1-methyl ethanol ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 8.21-8.20 (m, 2H), 7.65-7.64 (m, 2H), 7.54-7.50(m, 1H), 7.35-7.30 (m, 2H), 7.23-7.18 (m, 3H), 6.25-6.19 (m, 1H),5.99-5.85 (m, 1H), 4.48-4.37 (m, 2H), 4.23-4.14 (m, 1H), 4.09-4.04 (m,1H), 3.91-3.84 (m, 4H), 1.74-1.73 (m, 6H), 1.32-1.26 (m, 3H).

Referring to the method for producing the compound 001, phenol,L-alanine-1-(3-methyl-2-nitro-3H-imidazole-4-yl) ethanol esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.58-7.53 (m, 1H), 7.40-7.33(m, 2H), 7.30-7.22 (m, 3H), 7.22-7.20 (m, 1H), 6.26-6.23 (m, 1H),6.02-5.90 (m, 1H), 5.85-5.75 (m, 1H), 4.59-4.41 (m, 2H), 4.20-4.30 (m,1H), 3.97-3.90 (m, 4H), 1.60-1.59 (m, 3H), 1.34-1.27 (m, 3H).

Referring to the method for producing the compound 001, phenol,L-alanine-1-(5-nitro-furan-2-yl) ethanol ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 7.69 (d, 1H), 7.57-7.52 (m, 1H), 7.38-7.33 (m,2H), 7.26-7.19 (m, 3H), 6.93 (d, 1H), 6.27-6.21 (m, 1H), 5.97-5.90 (m,1H), 4.53-4.31 (m, 2H), 4.25-4.18 (m, 1H), 4.12-4.07 (m, 1H), 3.94-3.87(m, 1H), 1.57-1.56 (m, 3H), 1.35-1.29 (m, 3H).

Referring to the method for producing the compound 001, phenol,L-alanine-1-(5-nitro-thiophene-2-yl) ethanol ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 8.75 (s, 1H), 7.72 (s, 1H), 7.55-7.50 (m, 1H),7.36-7.31 (m, 2H), 7.24-7.18 (m, 3H), 6.26-6.21 (m, 1H), 6.07-6.01 (m,1H), 5.90-5.81 (m, 1H), 4.51-4.30 (m, 2H), 4.24-4.16 (m, 1H), 4.10-4.04(m, 1H), 3.92-3.86 (m, 1H), 1.60-1.59 (m, 3H), 1.32-1.27 (m, 3H).

Referring to the method for producing the compound 001,1-(4-nitrophenyl) ethanol, L-alanine benzyl ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 8.20-8.18 (m, 2H), 7.64-7.62 (m, 2H), 7.58-7.53(m, 1H), 7.39-7.34 (m, 5H), 6.28-6.23 (m, 1H), 5.92-5.76 (m, 2H),5.16-5.10 (m, 2H), 4.54-4.32 (m, 2H), 4.26-4.20 (m, 1H), 4.12-4.06 (m,1H), 3.95-3.90 (m, 1H), 1.56-1.53 (m, 3H), 1.35-1.30 (m, 3H).

Referring to the method for producing the compound 001,1-(4-nitrophenyl) ethanol, L-alanine methyl ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 8.22-8.20 (m, 2H), 7.67-7.65 (m, 2H), 7.60-7.55(m, 1H), 6.31-6.26 (m, 1H), 5.96-5.80 (m, 2H), 4.57-4.35 (m, 2H),4.29-4.23 (m, 1H), 4.15-4.09 (m, 1H), 3.97-3.92 (m, 1H), 3.58 (s, 3H),1.61-1.55 (m, 3H), 1.38-1.33 (m, 3H).

Referring to the method for producing the compound 001,1-(4-nitrophenyl) ethanol, L-alanine ethyl ester hydrochloride,gemcitabine and other raw materials were used for synthesis. 1H NMR(MeOD, 300 MHz) δ(ppm): 8.19-8.17 (m, 2H), 7.63-7.61 (m, 2H), 7.58-7.52(m, 1H), 6.26-6.21 (m, 1H), 5.90-5.75 (m, 2H), 4.52-4.30 (m, 2H),4.24-4.18 (m, 1H), 4.14-4.05 (m, 1H), 3.91-3.75 (m, 3H), 1.55-1.50 (m,3H), 1.34-1.29 (m, 3H), 1.16-1.12 (m, 3H).

Referring to the method for producing the compound 001,1-(4-nitrophenyl) ethanol, L-alanine isopropyl ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 8.21-8.19 (m, 2H), 7.65-7.63 (m, 2H), 7.60-7.56(m, 1H), 6.29-6.25 (m, 1H), 5.94-5.88 (m, 2H), 5.03-4.97 (m, 1H),4.55-4.34 (m, 2H), 4.28-4.22 (m, 1H), 4.15-4.10 (m, 1H), 3.98-3.93 (m,1H), 1.58-1.54 (m, 3H), 1.38-1.31 (m, 3H), 1.26-1.23 (m, 6H).

Referring to the method for producing the compound 001,1-(4-nitrophenyl) ethanol, L-alanine butyl ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ^(1H) NMR(MeOD, 300 MHz) δ(ppm): 8.23-8.21 (m, 2H), 7.65-7.63 (m, 2H), 7.62-7.57(m, 1H), 6.31-6.25 (m, 1H), 5.97-5.79 (m, 2H), 4.58-4.35 (m, 2H),4.30-4.24 (m, 1H), 4.17-4.12 (m, 1H), 4.00-3.95 (m, 1H), 3.88-3.78 (m,2H), 1.60-1.20 (m, 10H), 0.85-0.81 (m, 3H).

Referring to the method for producing the compound 001,1-(4-nitrophenyl) ethanol, L-alanine 2,2-dimethyl propyl esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ^(IH) NMR (MeOD, 300 MHz) δ(ppm): 8.24-8.22 (m, 2H),7.66-7.64 (m, 2H), 7.62-7.58 (m, 1H), 6.32-6.27 (m, 1H), 5.99-5.80 (m,1H), 4.60-4.38 (m, 2H), 4.32-4.26 (m, 1H), 4.18-4.14 (m, 1H), 4.02-3.96(m, 1H), 3.90-3.80 (m, 2H), 1.62-1.58 (m, 3H), 1.39-1.33 (m, 3H),1.24-1.21 (m, 9H).

Referring to the method for producing the compound 001,1-(4-nitrophenyl) ethanol, L-alanine cyclohexyl ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 8.19-8.17 (m, 2H), 7.63-7.61 (m, 2H), 7.57-7.52(m, 1H), 6.28-6.23 (m, 1H), 5.94-5.78 (m, 2H), 4.55-4.35 (m, 2H),4.28-4.20 (m, 2H), 4.15-4.10 (m, 1H), 4.02-3.94 (m, 1H), 1.70-1.52 (m,5H), 1.39-1.15 (m, 1l 1H).

Referring to the method for producing the compound 001,1-(5-nitrothiophene-2-yl) methanol, L-alanine cyclohexyl esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.08-8.07 (m, 1H), 7.65-7.55(m, 1H), 7.32-7.31 (m, 1H), 6.30-6.25 (m, 1H), 5.98-5.88 (m, 1H),5.25-5.07 (m, 3H), 4.55-4.35 (m, 1H), 4.28-4.21 (m, 1H), 4.15-4.10 (m,1H), 4.04-3.97 (m, 1H), 1.72-1.55 (m, 5H), 1.42-1.18 (m, 11H).

Referring to the method for producing the compound 001, 4-nitrobenzylalcohol, L-alanine methyl ester hydrochloride, gemcitabine and other rawmaterials were used for synthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm):8.20-8.19 (m, 2H), 7.65-7.63 (m, 2H), 7.59-7.53 (m, 1H), 6.28-6.24 (m,1H), 5.95-5.90 (m, 1H), 5.14-5.02 (m, 2H), 4.55-4.35 (m, 2H), 4.28-4.21(m, 1H), 4.15-4.10 (m, 1H), 3.98-3.90 (m, 1H), 3.56 (s, 3H), 1.57-1.53(m, 3H), 1.36-1.30 (m, 3H).

Referring to the method for producing the compound 001,1-(5-nitrofuran-2-yl) methanol, L-alanine benzyl ester hydrochloride,gemcitabine and other raw materials were used for synthesis. ¹H NMR(MeOD, 300 MHz) δ(ppm): 7.69-7.68 (m, 1H), 7.65-7.64 (m, 1H), 7.59-7.54(m, 1H), 7.36-7.29 (m, 5H), 6.29-6.24 (m, 1H), 5.94-5.88 (m, 1H),5.08-5.03 (m, 2H), 4.57-4.36 (m, 2H), 4.30-4.20 (m, 1H), 4.14-4.09 (m,1H), 3.97-3.88 (m, 1H), 3.89-3.60 (m, 2H), 1.56-1.51 (m, 3H), 1.35-1.31(m, 3H).

Referring to the method for producing the compound 001,1-(5-nitrothiophene-2-yl) methanol, L-alanine isopropyl esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.07-8.06 (m, 2H), 7.67-7.55(m, 1H), 7.31-7.300, 1H), 6.30-6.25 (m, 1H), 5.96-5.88 (m, 1H),5.22-5.18 (m, 2H), 5.09-4.98 (m, 1H), 4.56-4.35 (m, 1H), 4.32-4.21 (m,1H), 4.15-4.10 (m, 1H), 3.99-3.90 (m, 1H), 1.38-1.33 (m, 3H) 1.26-1.24(m, 6H).

Referring to the method for producing the compound 001,(3-methyl-2-nitro-3H-imidazole-4-yl) methanol, L-alanine ethyl esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.66-7.55 (m, 1H), 7.22-7.21(m, 1H), 6.29-6.23 (m, 1H), 5.94-5.86 (m, 1H), 4.54-4.31 (m, 2H),4.26-4.18 (m, 1H), 4.17-4.10 (m, 3H), 3.96-3.70 (m, 3H), 1.36-1.32 (m,3H), 1.16-1.13 (m, 3H).

Referring to the method for producing the compound 001, 4-methoxybenzylalcohol, L-alanine-1-methyl-1-(5-nitrofuran-2-yl) ethanol esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.68-7.66 (m, 1H), 7.61-7.56(m, 1H), 7.05-7.04 (m, 2H), 6.96-6.94 (m, 2H), 6.32-6.25 (m, 1H),5.96-5.87 (m, 2H), 5.10-5.03 (m, 2H), 4.57-4.34 (m, 2H), 4.29-4.21 (m,1H), 4.15-4.09 (m, 1H), 3.98-3.91 (m, 4H), 1.56-1.55 (m, 3H), 1.37-1.32(m, 3H).

Referring to the method for producing the compound 001, 2-methyl benzylalcohol, L-alanine-1-methyl-1-(5-nitrothiophene-2-yl) ethanol esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.73-8.72 (m, 1H), 7.64-7.63(m, 1H), 7.59-7.54 (m, 1H), 7.37-7.32 (m, 1H), 7.24-7.15 (m, 3H),6.31-6.23 (m, 1H), 5.95-5.89 (m, 1H), 5.13-5.00 (m, 2H), 4.56-4.32 (m,2H), 4.28-4.10 (m, 3H), 4.14-4.06 (m, 1H), 3.97-3.90 (m, 1H), 2.37-2.35(m, 3H), 1.82-1.81 (m, 6H), 1.37-1.30 (m, 6H).

Referring to the method for producing the compound 001,4-N,N-dimethylbenzyl alcohol,L-alanine-1-methyl-1-(5-nitrothiophene-2-yl) ethanol esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 8.20-8.18 (m, 2H), 7.61-7.56(m, 1H), 7.50-7.49 (m, 2H), 7.06-6.83 (m, 4H), 6.30-6.21 (m, 1H),6.02-5.89 (m, 2H), 5.11-4.98 (m, 2H), 4.56-4.32 (m, 2H), 4.29-419 (m,1H), 4.12-4.06 (m, 1H), 3.96-3.88 (m, 1H), 2.81-2.80 (m, 6H), 1.82-1.81(m, 6H), 1.35-1.29 (m, 3H).

Referring to the method for producing the compound 001, 2-methoxybenzylalcohol, L-alanine-1-(3-methyl-2-nitro-3H-imidazole-4-yl) ethanol esterhydrochloride, gemcitabine and other raw materials were used forsynthesis. ¹H NMR (MeOD, 300 MHz) δ(ppm): 7.55-7.61 (m, 1H), 7.35-7.25(m, 2H), 7.00-6.92 (m, 2H), 7.20-7.19 (m, 1H), 6.26-6.23 (m, 1H),5.96-5.84 (m, 1H), 5.04-4.90 (m, 2H), 4.58-4.40 (m, 2H), 4.28-4.17 (m,1H), 3.95-3.88 (m, 4H), 1.59-1.58 (m, 3H), 1.30-1.20 (m, 3H).

Embodiment 2: Research on In-Vitro Inhibitory Effect of Target Compoundon Tumor Cell Proliferation Under Normal Oxygen State and Hypoxic State

Take tumor cells in the logarithmic growth phase, add 0.25% pancreatinfor digestion for 3 min, use RPMI-1640 containing 10% calf serum forsuspension culture of the cells, count the number, adjust cellconcentration to 1×10⁵ cells/mL, inoculate to a Top-count dedicated96-well cell culture plate at 100 μL/well, and incubate at 37° C. and 5%CO₂ for 24 h; divide the cells into experimental groups and controlgroups, and add a target compound solution (0.001 μg/mL, 0.01 μg/mL, 0.1μg/mL, 1 μg/mL, 10 μg/mL) to the experimental groups, wherein eachconcentration corresponded to four wells, and the volume of each wellwas made up to 200 μL; after adding samples, continue to culture for 72h for each group (for hypoxic groups, continue to culture for 72 h at 5%CO₂, 95% N₂), add ³H-TdR 3×10⁵Bq to each group before culture ended, andmeasure the CPM (count per minute) value of each well with Top-count;and calculate the median inhibition concentration (IC₅₀) of drugs ineach experimental group on cell proliferation.

TABLE 1 Median inhibition concentration (IC₅₀, μ/mL) of target compoundon tumor cell proliferation (72 h) under normal oxygen and hypoxicconditions IC₅₀ normal oxygen (air)/IC₅₀ hypoxic (nitrogen) Human lungHuman liver Human pancreatic Compound adenocarcinoma canceradenocarcinoma cell number cell A549 HepG2 cell BxPC-3 cell 001 1/0.05 >10/0.3 >10/0.2 002 2/0.1 >10/0.2 >10/0.4 003 Notmeasured >10/0.4 Not measured 004 Not measured Not measured >10/0.4 0052/0.1 Not measured Not measured 006 2/0.2 Not measured >10/0.4 007 Notmeasured >10/0.5 >10/0.4 008 Not measured >10/0.3 >10/0.4 009 2/0.2 Notmeasured >10/0.4 010 2/0.2 Not measured Not measured 011 2/0.2 Notmeasured Not measured 012 Not measured >10/0.5 >10/0.5 013 Not measuredNot measured >10/0.8 014 Not measured Not measured >10/0.7 015 Notmeasured Not measured >10/0.5 016 Not measured Not measured >10/0.4 017Not measured Not measured >10/0.8 018 Not measured Not measured >10/0.5019 Not measured Not measured >10/0.8 020 Not measured Notmeasured >10/0.4 021 Not measured Not measured >10/0.5 022 0.01/0.01  0.4/0.3  0.1/0.1 023 0.01/0.01   0.4/0.3  0.1/0.1 024 Not measured Notmeasured  >10/>10 Gemcitabine 0.01/0.01   0.2/0.2  0.2/0.2 NUC-10310.01/0.01   0.2/0.2  0.1/0.1

The above experimental results show that gemcitabine, NUC-1031 andcompounds 022-024 have no significant difference in in-vitro inhibitionon tumor cell proliferation under normal oxygen and hypoxic conditions,and compounds (1-021) of the embodiments of the present invention havesignificant difference (10-50 times) in in-vitro inhibition on tumorcell proliferation under normal oxygen and hypoxic conditions,indicating that the compounds of the embodiments of the presentinvention have a stronger cytotoxicity for tumors in hypoxic regions.

Embodiment 3: Growth Inhibition Effect of Target Compound on OrthotopicTransplantation Tumor of Human BxPC-3 Nude Mice

Take BxPC-3 human pancreatic cancer cells in the logarithmic growthphase, inoculate subcutaneously on the back of nude mice at aconcentration of 5×10⁶ cells·0.2 mL⁻¹·mouse⁻¹, establish a human BxPC-3nude mice subcutaneous transplantation tumor model, take out aftergrowing into a 1 cm subcutaneous transplantation tumor, remove thecentral necrotic tissue under an aseptic condition, and select and cutthe surrounding healthy tumor tissue into 1 mm³ tissue blocks.

Preparation of surgical orthotopic transplantation model:intraperitoneally anesthetize nude mice with pentobarbital sodium (50mg/Kg), make a cut beside the left upper rectus abdominis muscle toexpose spleen and tail of pancreas, cut open capsula pancreatis, implanta tumor block into the tail of pancreas near splenic artery, and suturethe capsula pancreatis.

Administration scheme: model animals were randomly divided into anexperimental group (compound 001), a control group, a gemcitabine groupand an NUC-1031 group 3 weeks after operation, from the third week afteroperation, the nude mice were injected intraperitoneally (0.2 mmol/kg,twice per week) for 4 weeks, the nude mice were killed one week afterdrug withdrawal, and pancreatic tumor tissues were taken and weighed.See FIG. 1 for inhibition effect: growth inhibition effect of targetcompound on orthotopic transplantation tumor of human BxPC-3 nude mice.After administration, the pancreatic tumor tissue quality of nude micein the experimental group (compound 001) was significantly lower thanthose of the gemcitabine group and the NUC-1031 group, indicating abetter tumor growth inhibition effect.

Embodiment 4: Growth Inhibition Effect of Target Compound onSubcutaneous Transplantation Tumor of Human BxPC-3 Nude Mice

Take BxPC-3 human pancreatic cancer cells in the logarithmic growthphase, inoculate subcutaneously on the back of nude mice at aconcentration of 5×10⁶ cells·0.2 mL⁻¹·mouse⁻¹, three weeks later, afterthe long diameters of the transplanted tumors in nude mice were all ≥5mm, calculate the similar volume of tumor bodies based on the longdiameter and short diameter of the transplanted tumors. Nude mice weredivided into 5 groups by a random block design and allocation methodaccording to the tumor volume.

Administration scheme: 50 model animals were randomly divided into anegative control group, a low-dose group (compound 001, 0.1 mmol/kg), ahigh-dose group (compound 001, 0.4 mmol/kg), a gemcitabine hydrochloridegroup (0.2 mmol/kg), and a combined administration group (compound 001,0.1 mmol/kg+ gemcitabine hydrochloride, 0.1 mmol/kg), intraperitoneallyinjected (twice per week) for 3 weeks, and killed one week after drugwithdrawal. At the same time, animal weight was measured and the eyecondition of the animal was observed.

See FIG. 2 for inhibition effect and FIG. 3 for weight change: afteradministration, each group showed a significant tumor growth inhibitioneffect, and the high-dose group and the combined administration groupshowed a better therapeutic effect. The weights of nude mice in allexperimental groups had no significant difference, but were smaller thanthose of the control groups.

The above examples are only to illustrate the technical concept andfeatures of the present invention, with the purpose of enabling thosefamiliar with the technology to understand the content of the presentinvention and implement it accordingly, but do not limit the scope ofprotection of the present invention. All equivalent changes ormodifications made in accordance with the spirit of the presentinvention should be included within the scope of protection of thepresent invention.

What is claimed is:
 1. A gemcitabine ProTide hypoxic-activated prodrug,wherein the chemical structural formula of the gemcitabine ProTidehypoxic-activated prodrug is:

wherein one of R¹ and R² is a hypoxic-activated group of —C(R³R⁴)ArNO₂,the other of R¹ and R² is an alkyl group of 1 to 6 carbon atoms, aphenyl group or —CH₂Ar, R³ and R⁴ are —H or a methyl group, and —Ar isan aromatic ring compound.
 2. The gemcitabine ProTide hypoxic-activatedprodrug according to claim 1, wherein the structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ is —H or amethyl group, and R⁴ is a methyl group.
 3. The gemcitabine ProTidehypoxic-activated prodrug according to claim 1, wherein R¹ is a phenylgroup, the structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 4. The gemcitabineProTide hypoxic-activated prodrug according to claim 1, wherein thestructure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, and R³ and R⁴ are—H.
 5. The gemcitabine ProTide hypoxic-activated prodrug according toclaim 1, wherein R¹ is —CH₂Ar, —Ar is a benzene ring with an electrondonating group, the structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 6. The gemcitabineProTide hypoxic-activated prodrug according to claim 1, wherein thechemical structural formula of the gemcitabine ProTide hypoxic-activatedprodrug is one selected from the group consisting of chemical structuralformulas as follows:


7. The gemcitabine ProTide hypoxic-activated prodrug according to claim1, wherein the chemical structural formula of the gemcitabine ProTidehypoxic-activated prodrug is one selected from the group consisting ofchemical structural formulas as follows:


8. A medicament for treating tumors, wherein an effective component ofthe medicament for treating tumors is the gemcitabine ProTidehypoxic-activated prodrug according to claim 1 or pharmaceuticallyacceptable salt of the gemcitabine ProTide hypoxic-activated prodrug. 9.The medicament according to claim 8, wherein the structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, R³ is —H or amethyl group, and R⁴ is a methyl group.
 10. The medicament according toclaim 8, wherein R¹ is a phenyl group, the structure of R² is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 11. The medicamentaccording to claim 8, wherein the structure of R¹ is:

R² is an alkyl or benzyl group of 1 to 6 carbon atoms, and R³ and R⁴ are—H.
 12. The medicament according to claim 8, wherein R¹ is —CH₂Ar, —Aris a benzene ring with an electron donating group, the structure of R²is:

R³ is —H or a methyl group, and R⁴ is a methyl group.
 13. The medicamentaccording to claim 8, wherein the chemical structural formula of thegemcitabine ProTide hypoxic-activated prodrug is one selected from thegroup consisting of chemical structural formulas as follows:


14. The medicament according to claim 8, wherein the chemical structuralformula of the gemcitabine ProTide hypoxic-activated prodrug is oneselected from the group consisting of chemical structural formulas asfollows: